The present study was undertaken to define the role of L-arginine (L-Arg) in glucose metabolism in differentiated 3T3-L1 adipocytes in culture. L-Arg alone had no effect on 2-deoxyglucose uptake or basal glycogen synthesis, but this amino acid increased by 153+10% (p<0.01) the incorporation of glucose into glycogen in insulin-treated cells. L-Glutamate (L-Glu), a major metabolite of L-Arg, also enhanced insulin-stimulated glycogen synthesis. The response to insulin was not altered by L-lysine (L-Lys), but the effect of L-Arg was markedly attenuated by L-Lys. Cell incubation with L-Arg markedly enhanced arginase-mediated urea synthesis while L-Lys abolished this response. The stimulatory effect of L-Arg on insulin-stimulated glycogen synthesis did not appear to be accounted for by the generation of polyamines or the production of nitric oxide, both potentially derived from the enzymatic conversion of L-Arg. In the presence of insulin, cellular ATP levels were significantly increased by L-Arg, L-Glu and L-Lys as well. These data suggest that metabolic degradation of L-Arg not related to citric acid cycle activity is important in the mechanism by which L-Arg enhances insulin-stimulated glycogen synthesis.